Toll-like Receptor 7/8 (TLR7/8)

Resiquimod (R-848) 会导致半抗原和过敏原特异性的循环 T 细胞（包括 TH2 效应细胞）产生 IFN-γ，甚至失去产生 IL-4 的能力 [2]。在 BrdU 掺入实验中，瑞西莫德 (R848) 增加了 BrdU 阳性细胞的数量，并以剂量依赖性方式促进 PBL 增殖。在用 R848 处理的细胞中，NF-κB 活性的报告者荧光素酶显着增加（3.5 倍）[3]。

在动物模型中，大鼠和小鼠可用作瑞西莫德免疫介导的心脏组织损伤和细胞因子产生的模型。在 SPF 鸡中，肌肉注射 Resiquimod (R-848)，剂量为 50 μg/只，可显着增加 IFN-α、IFN-β、IFN-γ、IL-1β、IL-4、iNOS 和 IFN-α 的产生。 MHC-II 基因 [1]。

BrdU掺入测定法[3]
对于BrdU掺入测定，R848（1 µg/ml），CQ（10 µM）、CQ加R848或PBS加入到如前所述的6孔组织培养板中的PBL中（2 × 106 细胞/孔）。细胞在22 °C持续48 h、 并且根据制造商的说明书用BrdU细胞增殖测定试剂盒检测细胞增殖。测定进行了三次。

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萤光素酶报告基因测定法[3]
对于萤光素酶测定，用萤火虫NF-κB-特异性萤光素酶报告载体pNFκB-Met-Luc2（Clontech，Mountain View，CA，USA）转染FG-9307细胞，如前所述（Chi等人，2013）。pNFκB-MetLuc2被设计用于直接从细胞培养基中监测NF-κB信号转导途径的激活。该载体含有位于最小TA启动子（PTA）上游的NF-κB增强子元件。位于PTA下游的是一个分泌的萤光素酶基因。转录因子与NF-κB增强子元件的结合允许MetLuc表达并分泌到周围的培养基中。pNFκB-MetLuc2已被证明在鱼类系统中起作用（Lauksund等人，2009）。通过与pSEAP2对照载体共转染来监测转染效率，该载体组成性地表达人分泌的增强型碱性磷酸酶（SEAP）。然后用R848（1 µg/ml），CQ（10 µM）、CQ加R848或PBS，并在22 °C持续24 h.然后分别使用萤光素酶测定试剂盒和Great EscAPe™SEAP化学发光检测试剂盒分析转染物的培养基的萤光素酶活性和SEAP活性。测定进行了三次。

MTT法测定细胞增殖[3]
为了制备PBL，从比目鱼尾静脉采集血液，并用L-15培养基以1:5稀释。将稀释后的血液放在61%Percoll上，并在400下离心 × 30的g 分钟。回收PBL层并用PBS洗涤三次。将细胞分布在96孔组织培养板（5 × 105 细胞/孔）在含有10%胎牛血清（FBS）的L-15培养基中，100 U/ml青霉素和100 µg/ml链霉素。用不同浓度（0、0.175、0.25、0.5、1、2、4、8和16 µg/ml）的R848用于48 h.为了抑制溶酶体酸化，将细胞与10 µM CQ用于1 R848处理前h。治疗后，20 µl，共5个 将mg/ml MTT{3-（4,5）-二甲基噻嗪（-z-y1）-3,5-二-苯并四唑溴化}加入到板中。培养板在22 °C 4 h、 和200 向板中加入µl二甲基亚砜以溶解还原的甲zan。然后在490读取铭牌 nm的微板读数器。为了确定Myd88抑制对R848诱导的细胞增殖的影响，将Myd88抑制剂Pepinh MYD（RQIKIWFQNRRMKWKK-RDVLPGTCVNS-NH2）和对照肽Pepinh control（RQIKiwFQNRRMKW KK-SLHGRGDPMEAFII-NH2）以50 µM，并将板在22 °C持续6 h.孵育后，用R848处理细胞，并如上所述进行MTT测定。为了确定NF-κB失活对R848诱导的细胞增殖的影响，将BAY-11-7082，一种IκB-α磷酸化的不可逆抑制剂，以1 µM，并将板在22 °C 1 h.孵育后，用R848处理细胞，并如前所述进行MTT测定。所有实验都进行了三次。

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凋亡检测[3]
将先前制备的PBL分布在12孔组织培养板中（1 × 106 细胞/孔）在含有10%FBS的L-15培养基中，100 U/ml青霉素和100 µg/ml链霉素。R848（1 µg/ml），CQ（10 µM）、CQ加R848或PBS加入细胞中，并在22 °C持续12 h或48 h.孵育后，用冷PBS洗涤细胞两次。根据制造商的说明，使用结合了FITC的膜联蛋白V-FITC和PI细胞凋亡检测试剂盒，用FITC结合的膜联素V和碘化丙啶（PI）处理洗涤的细胞。然后使用配备有FlowJo软件（Tree Star Inc，Ashland OR）的FACSort流式细胞仪对细胞进行流式细胞术以进行数据分析。为了确定Myd88抑制对细胞凋亡的影响，将Pepinh MYD和Pepinh对照以50 µM。22孵化后 °C持续6 h、 用R848处理细胞，并如前所述进行膜联蛋白V/PI测定。为了确定NF-κB失活对R848诱导的抗细胞凋亡的影响，将BAY-11-7082以1 µM。22℃孵育后 °C 1 h、 用R848处理细胞，并如前所述进行膜联蛋白V/PI测定。所有实验都进行了三次。

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定量实时逆转录聚合酶链式反应（qRT-PCR）[3]
用RNAprep组织试剂盒从PBL中提取总RNA。使用M-MLV逆转录酶将1微克总RNA用于cDNA合成。如前所述（Zheng和Sun，2011），在Eppendorf Mastercycler中使用SYBR ExScript qRT-PCR试剂盒进行qRT-PCR。在每次PCR结束时进行扩增产物的熔解曲线分析，以确认仅扩增并检测到一种PCR产物。以β-肌动蛋白为内参照，采用比较阈值循环法分析靶基因的表达水平（Zhang et al.，2013）。实验进行了三次。

R848 and chloroquine (CQ) were resuspended in PBS to 200 µg/ml and 100 µM respectively. Japanese flounder (average 11.6 g) were divided randomly into four groups and administered by intramuscular (i.m.) injection with 50 µl R848, CQ, R848 plus CQ, or PBS. At 24 h post-administration, the fish were challenged by intraperitoneal (i.p.) injection with 50 µl megalocytivirus that had been suspended in PBS to 1 × 107 copies/ml. At 3 d, 5 d, and 7 d post-challenge, kidney and spleen were collected from the fish (4 fish/time point), and the viral amounts in the tissues were determined by absolute quantitative real time PCR as reported previously (Zhang et al., 2012). The experiment was performed three times[3].

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Although the solitary adjuvant potential of R-848 is well established in mammals, such reports are not available in avian species hitherto. Hence, the adjuvant potential of R-848 was tested in SPF chicken in this study. Two week old chicks were divided into four groups (10 birds/group) viz., control (A), inactivated Newcastle disease virus (NDV) vaccine prepared from velogenic strain (B), commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain (C) and inactivated NDV vaccine prepared from velogenic strain with R-848 (D). Booster was given two weeks post primary vaccination. Humoral immune response was assessed by haemagglutination inhibition (HI) test and ELISA while the cellular immune response was quantified by lymphocyte transformation test (LTT) and flow cytometry post-vaccination. Entire experiment was repeated twice to check the reproducibility. Highest HI titre was observed in group D at post booster weeks 1 and 2 that corresponds to mean log2 HI titre of 6.4 ± 0.16 and 6.8 ± 0.13, respectively. The response was significantly higher than that of group B or C (P<0.01). LTT stimulation index (P ≤ 0.01) as well as CD4(+) and CD8(+) cells in flow cytometry (P<0.05) were significantly high and maximum in group D. Group D conferred complete protection against virulent NDV challenge, while it was only 80% in group B and C. To understand the effects of R-848, the kinetics of immune response genes in spleen were analyzed using quantitative real-time PCR after R-848 administration (50 μg/bird, i.m. route). Resiquimod significantly up-regulated the expression of IFN-α, IFN-β, IFN-γ, IL-1β, IL-4, iNOS and MHC-II genes (P<0.01). In conclusion, the study demonstrated the adjuvant potential of R-848 when co-administered with inactivated NDV vaccine in SPF chicken which is likely due to the up-regulation of immune response genes[1].

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Dissolved in saline; 50 nmol; i.p. injection

Wild-type mice, TLR7-deficient mice, and MyD88-deficient mice

[1]. Factors Influencing the Differentiation of Human Monocytic Myeloid-Derived Suppressor Cells Into Inflammatory Macrophages. Front Immunol. 2018 Mar 26;9:608.

[2]. FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis. Cells. 2022 Apr 14;11(8):1341.

[3]. Resiquimod, a TLR7/8 agonist, promotes differentiation of myeloid-derived suppressor cells into macrophages and dendritic cells. Arch Pharm Res. 2014;37(9):1234-40.

Description
Resiquimod is an imidazoquinoline.
A substance being studied in the treatment of some types of skin cancer. When put on the skin, resiquimod causes some immune cells to make certain chemicals that may help them kill tumor cells. It is also being studied to find out if adding it to a tumor vaccine improves the antitumor immune response. It is a type of imidazoquinoline and a type of immunomodulator.
Resiquimod is an imidazoquinolinamine and Toll-like receptor (TLR) agonist with potential immune response modifying activity. Resiquimod exerts its effect through the TLR signaling pathway by binding to and activating TLR7 and 8 mainly on dendritic cells, macrophages, and B-lymphocytes. This induces the nuclear translocation of the transcription activator NF-kB as well as activation of other transcription factors. This may lead to an increase in mRNA levels and subsequent production of cytokines, especially interferon-alpha (INF-a) and other cytokines, thereby enhancing T-helper 1 (Th1) immune responses. In addition, topical application of resiquimod appears to activate Langerhans' cells, leading to an enhanced activation of T-lymphocytes. Due to its immunostimulatory activity, this agent may potentially be useful as a vaccine adjuvant.

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Drug Indication
Investigated for use/treatment in genital herpes.

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Mechanism of Action
Resiquimod is a Toll-like receptor 7 (TLR7) and TLR8 agonist that is a potent inducer of alpha interferon (IFN-alpha) and other cytokines.

C17H22N4O2

314.38

314.174

C, 64.95; H, 7.05; N, 17.82; O, 10.18

144875-48-9

Resiquimod-d5;2252319-44-9;Resiquimod;144875-48-9

159603

White to light yellow solid

1.3±0.1 g/cm3

553.6±50.0 °C at 760 mmHg

193 °C

288.6±30.1 °C

0.0±1.6 mmHg at 25°C

1.635

2.15

86.19

2

5

5

23

406

0

O([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])N1C(C([H])([H])OC([H])([H])C([H])([H])[H])=NC2C(N([H])[H])=NC3=C([H])C([H])=C([H])C([H])=C3C1=2

BXNMTOQRYBFHNZ-UHFFFAOYSA-N

InChI=1S/C17H22N4O2/c1-4-23-9-13-20-14-15(21(13)10-17(2,3)22)11-7-5-6-8-12(11)19-16(14)18/h5-8,22H,4,9-10H2,1-3H3,(H2,18,19)

1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.95 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (6.62 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (6.62 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 2.08 mg/mL (6.62 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，您可以将 100 μL 20.8 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。